1. Field of the Invention
The invention is generally related to recombinant deoxyribonucleic acid (DNA) technology and, more particularly, to cloning the genes responsible for biosynthesis of poly-beta-hydroxybutyrate (PHB) from Alcaligenes eutrophus H16 (A. eutrophus) into Escherichia coli (E. coli) and expressing the PHB biosynthetic pathway in E. coli.
2. Description of the Prior Art
PHB is an energy storage material produced by a variety of bacteria in response to environmental stress. Lemoigne discovered the presence of PHB in Bacillus in 1926 and it has since been identified in several different bacterial genera, including Azotobacter, Beijerinckia, Alcaligenes, Psuedonomas, Rhizobium, and Rhodospirillum. PHB is a homopolymer of D-(-)-3-hydroxybutyrate and has properties comparable to polypropylene. PHB is commercially produced using A. eutrophus and is sold on the market under the tradename Biopol. An exciting feature of PHB relative to other commercially available plastics is its biodegradability which makes it more suitable for packaging purposes since it will not have an adverse environmental impact. PHB has also been used as a source of chiral centers for the organic synthesis of certain antibiotics, and has been utilized in drug delivery and bone replacement applications.
The biosynthesis of PHB has been studied extensively in A. eutrophus and Azotobacter beijerinckii. FIG. 1 outlines a three step biosynthetic pathway for PHB found in most prokaryotic organisms. Beta-ketothiolase first catalyzes the reversible condensation of two acetyl coenzyme A (CoA) molecules to acetoacetyl-CoA. The acetoacetyl-CoA is reduced by acetoacetyl-CoA reductase to D-(-)-3-hydroxybutyryl-CoA. Enzyme action of the acetoacetyl-CoA reductase is dependent on NADPH. PHB synthetase polymerizes the D-(-)-3-hydroxybutyryl-CoA to PHB.
PHB accumulates when growth of a bacteria culture is restricted by a nutrient other than a carbon source. For example, oxygen deprivation, nitrogen deprivation, sulfate limitation and magnesium limitation have all been used as limitations on environmental conditions. Under such environmental conditions, the PHB content in bacteria cells can increase to as much as 80% of the dry weight. When the limiting conditions are relaxed, PHB quantities decrease to preinduction levels. Induction studies in which beta-ketothiolase and acetoacetyl CoA reductase were studied have revealed that both enzymatic activities increase markedly in response to PHB-stimulating limitation conditions.
Examples of U.S. Patents dealing with the biotechnological production of PHB and extraction of PHB from microorganisms include the following: U.S. Pat. No. 4,786,598 to Lafferty et al. discloses a two stage fermentation process where PHB is produced using Alcaligenes latus, U.S. Pat. No. 4,705,604 to Vanlautem et al. discloses using 1,2 dichloroethane to simultaneously remove water from the bacterial suspension by azeotropic distillation and extract PHB from the cells, U.S. Pat. No. 4,477,654 to Holmes et al discloses limiting the nitrogen nutrient source to microbiologically accumulate 3-hydroxybutyrate polymers, U.S. Pat. No. 4,433,053 discloses a fermenting process for PHB accumulation using A. eutrophus where a nutrient required for growth is limited, U.S. Pat. No. 4,336,334 to Powell et al. shows a microbiological process for producing PHB using Methylobacterium organophilum, U.S. Pat. No. 4,358,583 to Walker et al. discloses extracting PHB by first flocculating the cells by heat or pH treatment then extracting with a suitable solvent, U.S. Pat. No. 4,138,291 to Lafferty discloses bacterial strains assimilating various carbon sources and converting them to PHB, and U.S. Pat. No. 3,121,669 to Baptist shows adding acetic acid vapor to an aerated stream of culture medium for production of PHB.
Although PHB can be produced in large amounts in natural bacterial genera according to the techniques described above, these bacteria are less manipulatable and not as well characterized as E. coli. In the field of genetic engineering, a relatively large body of knowledge exists for E. coli. E. coli have been utilized as host cells for producing a Wide variety of products including Human Growth Hormone, insulin and interferon. In order to make PHB production more regulatable, a need exists for cloning the PHB biosynthetic pathway into E. coli.